Two canonical binding sites for the Ets family of transcription factors have been identified within the TCR-zeta promoter. These sites bind Elf- 1, and play an important role in basal transcription of zeta. Ligand- mediated TCR degradation is inhibited by reagents that block vesicular acidification but not substantially affected by inhibitors of the ubiquitin/proteasome pathway, suggesting that lysosomal degradation is the major route leading to loss of cell surface receptors. However, we have now determined that the degradation of unassembled TCR subunits from the ER does involve proteasomal degradation and the generation of ubiquitinated intermediates, and we are in the process of characerizing this process further. Our determination that TCR subunits are substrates for ubiquitination has led us to evaluate ubiquitination in more detail. A family of human E2 enzymes (UbcH5A-C) have been cloned and characterized. These E2s are widely expressed in human tissues and function in conjunction with E6-AP, in the ubiquitination of proteins. We have also been studying a novel yeast E2, UBC6, a novel type IV transmembrane protein found associated with membranes of the ER. The short transmembrane domain of UBC6 is responsible for its distribution within the cells, progressive lengthening the transmembrane segment re- targets UBC6 to the Golgi Apparatus and then to the cell surface. More recently we have determined the existence of a mammalian UBC6 homolog, which we are in the process of characterizing. We have also recently characerized two murine E3 enzymes. One of these is the murine homolog of E6-AP, the other is a protein found to have homology to E6-AP, termed Nedd-4. Like E6-AP, Nedd-4 is an E3 enzyme, both of these function with the UbcH5 family of E2s in the ubiquitination of cellular proteins. These E3s exhibit distinct subcellular localizations, E6-AP is expressed predominantly in the nucleus, Nedd-4 is present exclusively in the cytosol. Nedd-4 has two distinct E2 bindings sites, one of which binds the amino terminus of E2s and the other shows affinity solely for the carboxyl component of E2s. These data suggest models for ubiquitination involving oligomerization of E2 and E3s. We are currently in the process of identifying binding partners for these E2s and E3s using the yeast two hybrid approach.